Vaccines against tick-borne encephalitis viruses. Tick-borne encephalitis viruses are important neurotropic human pathogens, causing a devastating and often fatal neuroinfection. A live attenuated TBEV vaccine is expected to induce a more durable protective immunity than that induced by the inactivated TBEV vaccine that is not licensed in the USA. The live attenuated TBEV vaccine candidates are being developed in the LID using a strategy based on chimerization of a neurovirulent TBEV with a non-neuroinvasive, mosquito-borne dengue-4 virus (DEN4d30) that contains an attenuating mutation, a 30 nucleotide deletion in the 3non-coding region. In FY 2009, we have focused on the identification of a chimeric TBEV/DEN4 vaccine candidate that is suitable for evaluation in humans and that exhibits the following important properties: (1) reduced neurovirulence and restricted replication in the brains of mice;(2) restricted or ablated neuroinvasiveness in immunodeficient mice;(3) decreased replication in rhesus monkeys;(4) ability to induce a protective TBEV-specific immune response in monkeys;and (5) a low level of neurovirulence in the CNS of non-human primates. The results of histopathological analysis of the CNS together with clinical observations and data on chimeric TBEV/DEN4d30 virus replication in the brain and spinal cord of monkeys indicated that chimerization of a neurovirulent TBEV strain with non-neuroinvasive DEN4d30 had an attenuating effect on the neurovirulence of TBEV in monkeys. However, this effect was insufficient to justify bringing this vaccine candidate to trials in humans. Further attenuation of TBEV/DEN4d30 neurovirulence was undertaken by introducing amino acid substitutions that had previously been shown to reduce replication of tick-borne Langat virus (LGT) or DEN4 in suckling mouse brain. When two amino acid substitutions (Lys315 >Glu in the structural envelop E protein and Asp654Arg655 >AlaAla in the non-structural NS5 protein) were introduced into TBEV/DEN4d30 genome, the resulting virus (TBEV/DEN4d30-E315-NS5-654,655) demonstrated the desired properties of an acceptable live attenuated vaccine candidate as it displayed (1) a 490-fold decrease in neurovirulence in suckling mice, (2) more than 10,000-fold restriction of replication in the brain of suckling mice, (3) a non-neuroinvasive phenotype in immunodeficient mice, (4) was still safe and immunogenic in monkeys, and (5) failed to infect and replicate in mosquitoes and ticks. A seed TBEV/DEN4d30-E315-NS5-654,655 virus was recently generated, and its neurovirulence and protective efficacy in non-human primates should be evaluated before clinical trials can be initiated. Since TBEV/DEN4d30 evoked a strong cellular inflammatory response in the CNS of monkeys, we have developed a new computerized method for quantitative evaluation of the CNS infiltration with peripheral immune cells (CD3, CD4, CD8, and CD20 lymphocytes) and the response of CNS resident cells (microglial activation and neuronal degeneration). We found that immunoreactivity for CD3 T cells and CD20 B cells correlated remarkably well with semi-quantitative histopathological scores for cellular inflammatory infiltration in the CNS of monkeys inoculated with either yellow fever (YF) 17D, LGT or TBEV/DEN4d30 attenuated viruses. Our data indicate that a high CD4:CD8 T cell ratio in the CNS inflammatory infiltrates induced in response to YF 17D infection is a major factor that differentiates this successful vaccine from a more neurovirulent LGT or TBEV/DEN4d30 virus. It is likely that the balanced response of T and B cells within the CNS of monkeys induced with YF 17D vaccine virus plays an important role in the recovery from CNS infection and might serve as a reference to evaluate the safety of new live flavivirus vaccine candidates. Vaccines against West Nile virus and St. Louis encephalitis virus. A live attenuated WN/DEN4d30 virus vaccine is being developed in the LID to protect humans against WN disease. In FY 2009, in a clinical trial in healthy adult volunteers, the WN/DEN4d30 vaccine was well tolerated, safe, and induced a potent and durable WN antibody response after two doses (100,000 PFU) of immunizations. Further studies of neurovirulence and neuropathogenesis in monkeys are necessary to evaluate the safety of this vaccine for the CNS of non-human primates prior to its use in the risk group of volunteers >50 years of age. Chimerization of SLE with DEN4 resulted in only moderate restriction in replication in rhesus monkeys, whereas the presence of the d30 mutation led to over-attenuation. Both chimeras (SLE/DEN4 and SLE/DEN4d30) retained high neurovirulence in suckling mice. In an effort to identify a SLE/DEN4 virus that will exhibit a satisfactory balance between attenuation and immunogenicity in mice and monkeys, we generated a set of SLE/DEN4 mutants by introducing the attenuating mutations such as the paired charge-to-alanine mutations in the NS5 protein and a Phe156>Ser mutation in E. Pre-clinical testing of these mutants for neurovirulence in mice and immunogenicity in monkeys will be initiated.